EsPIP5K4, a target of Es-miR408, positively regulates salt tolerance in Siberian wildrye.

Siberian wildrye (Elymus sibiricus L.) is a native grass of significant ecological and forage value for ecological restoration and livestock production in western China. However, the miRNA-mediated regulatory mechanisms underlying its salt tolerance remain largely unexplored. In this study, through integrative miRNA-mRNA profiling under salt stress, we identified EsPIP5K4 as a candidate target of Es-miR408. The inverse expression patterns of Es-miR408 (downregulated) and EsPIP5K4 (upregulated) under salt stress, together with dual-luciferase reporter assays, indicated that Es-miR408 negatively regulates EsPIP5K4 through its 3'UTR. Functional validation using TRV-mediated virus-induced gene silencing (VIGS) of EsPIP5K4 in Siberian wildrye significantly compromised salt tolerance, as evidenced by greater malondialdehyde (MDA) accumulation, reduced proline increase, and accelerated chlorophyll degradation under salt stress. Conversely, heterologous overexpression of EsPIP5K4 in tobacco conferred enhanced salt tolerance, accompanied by improved physiological performance and altered stomatal traits under salt stress. Collectively, our findings support a model in which salt stress downregulates Es-miR408, thereby relieving repression of EsPIP5K4 and contributing to salt tolerance. This study not only reveals a novel Es-miR408-EsPIP5K4 regulatory module for salt adaptation in Siberian wildrye, but also provides a potential genetic target for molecular breeding of salt-tolerant forage grasses.